This invention relates to a mutant mouse having a defect in an endogenous .alpha.4 subunit of the nicotinic acetylcholine receptor and to a transgenic animal based on the mutant mouse. In addition, this invention relates to isolated cells derived from these animals and to the use of the animals in assays for assessing the effects of nicotine and nicotinic agonists and antagonists in these animals and cells. Finally, this invention provides a nucleotide construct for use in generating these animals and cells.
The nicotinic acetylcholine receptor (nAChR) is a pentameric protein that forms a non-selective cation channel at the neuro-muscular junction and in the central nervous system (CNS). The genes encoding the neuronal nicotinic receptor subunits represent a large multigene family consisting of at least eight alpha subunits (.alpha.2-9) and three beta subunits (.beta.2-4). Each subunit has four putative transmembrane-spanning domains (M1-4) and a similar topological structure.
Neuronal receptors form either heteropentamers comprised of a total of 5 subunits: Two alpha subunits (.alpha.2, .alpha.3, .alpha.4, or .alpha.6) and three beta subunits (.beta.2 or .beta.4) or they are capable of forming homopentamers (.alpha.7, .alpha.8 or .alpha.9) (Refs. 1 and 2). The binding site for acetylcholine (ACh) is found at the interface between .alpha. and .beta. subunits indicating that both types of subunits contribute to the various pharmacological profiles of neuronal nAChRs (Ref. 3).
In situ hybridization experiments have shown the neuronal nAChR subunits to have diverse yet overlapping expression patterns in the CNS. .beta.2 is the most widely expressed subunit in the peripheral nervous system (PNS), while .alpha.4 expression is almost as ubiquitous, but .alpha.4 is more limited to the CNS with only a small amount of expression seen in the PNS (Refs. 4 and 5). These findings have been further confirmed by immunoprecipitation experiments, which indicate that in vivo receptors in the CNS are primarily composed of .alpha.4 and .beta.2 subunits (Ref. 6).
The pharmacology of the nAChRs has been extensively demonstrated with the Xenopus oocyte heterologous expression system (Ref. 7). This approach is limited, however, as the endogenous composition of nAChRs is still not known. While the rank order of potencies of nicotinic agonists seen in vivo more or less correspond to those observed in vitro, the single channel conductance of nAChR in vivo rarely coincides with the values found in Xenopus oocytes (Ref. 2).
Another approach to identify the composition of nAChR is to examine the nicotinic responses in knockout animals. This approach has been validated with the .beta.2 nAChR subunit knockout mice where the .beta.2 subunit was found to be necessary for a nicotine elicited response in the thalamus, but not in the medial habenula (Ref. 8). Furthermore, .beta.2 was found to be a necessary component of the high affinity binding site for nicotine.
Moreover, certain behavioral effects of systemic nicotine are not clearly associated with the activation of particular structures. Nicotine has been demonstrated to act as an enhancer of memory and attention (Ref. 10), as an antinociceptive (Ref. 11), and as an anxiolytic (Ref. 12). The composition of the subunits of the nAChRs that mediate these responses are not known. On the other hand, the activation of nAChR by endogenous ACh in these different behaviors remains to be demonstrated. Thus, there exists a need in the art for a way to examine this contribution by comparison with wild type animals.
Further, because the precise roles of the various subunits of the nAChR are unknown at this time, there exists a need in the art for an animal model to study such roles. The generation of a mutant mouse having defective nAChR subunits would aid in defining the normal roles of the various subunits, and allow an animal model of nAChR deficiency to be used in the design and assessment of chemical approaches to regulating nicotine effects. Such a nAChR modified mutant or transgenic animal could also be used as a source of cells for cell culture.